mimbfandomcom-20200214-history
Methods in Molecular Biosciences Wiki
Welcome This Wiki is intended to give beginning graduate students an overview of the methods commonly used in the many fields that are encompassed in the molecular biosciences. It is in no way intended to be a complete and thorough presentation of all molecular and cellular methods, rather it is presented as a guide to help students navigate the current literature and their work during rotations. It contains, in narrative fashion, brief descriptions of methods, usually with links to the corresponding wikipedia page, which will have much more detail. Free Advice We offer one very important piece of advice for beginning (and sage old) scientists. For every single experiment, even the most mundane, you MUST do a positive and negative control. Don’t allow yourself to pick up a pipet until you’ve planned a positive and negative control into the experiment - even for the simplest cloning experiments. The nature of these controls will vary greatly depending on the experiment, and some controls will be better than others, but they MUST be done. Narrative: p53 In order to make this wiki a bit more interesting, we are going to follow the expression and function of a single gene. In many respects, most of the questions asked in Molecular Bioscience labs are in some way focused on differential gene expression and the functional states that result from such. In that vein, we present a set of methods that could be employed to investigate the expression and function of the highly pluripotent p53 gene (TP53). GENE STRUCTURE Question: What is the structure of the TP53 gene? The first thing you might want to do is look at the gene’s structure. You can begin by visiting the NCBI home page , selecting the Gene database in the dropdown and doing a query for “human TP53.” Note that all human genes are annotated as all-cap italics and you should note that protein names and gene names often do not match - one must do some searching to make sure you make the correct association. The top hit will bring you to a page that contains a vast amount of information regarding p53. For now we are just interested in gene structure, so click on the bar titled “Genomic regions, transcripts, and products” and you will see all of the NCBI database entries that correspond to this gene. In playing with this interface you can see the number of introns and isoforms that are expressed. Some argue that the UCSC Genome Browser provides a better interface for gene searches - you be the judge. Question: How do you identify a p53 knockout mouse? 'One of the reagents you possess is a knockout mouse, but your technician mixed up cages. In order to determine which mice are p53 null, you must isolate DNA and verify that the gene has been deleted. There are two basic methods for DNA detection: 'PCR. By far the most common method for detection and amplification of specific DNA sequences, you use short specific single-stranded oligonucleotide “primers” that are complementary to the 5’ and 3’ ends of the fragment of DNA you want to detect. Mix these together with some purified DNA and a thermostable DNA polymerase (Taq), and you will amplify the sequence between your two primers. In the case of p53, you would choose primers that span all of the deleted exons so that the product obtained from p53 null mice is much smaller than the one obtained from wild-type mice. Southern Blot. Here you take your purified genomic DNA and cut it up with a restriction enzyme that will cut with reasonably low frequency in the genome (typically one that recognizes a 6-nucleotide sequence, e.g. EcoR1). You resolve your restriction fragments on an agarose gel, transfer those fragments to a piece of nitrocellulose membrane. You then generate a radioactive or fluorescently labeled “probe” that is a piece of DNA or RNA that is complementary to your target gene. Based on the known sequence of the gene and its predicted pattern of EcoR1 fragments, you can easily tell the difference between wild-type and p53 null mice. PHYLOGENETIC ANALYSIS Question: Is the p53 protein highly conserved across all three domains of life? Arguably the first thing you would want to do when considering a study of p53 (or any protein) structure and function is a phylogenetic analysis. A lot can be learned from finding out how many different types of organisms express p53 and which domains of the protein are conserved. For example, you will find that Drosophila expresses p53, but fruit flies don’t get cancer...so tumor suppression is probably not the evolutionarily linked basal function for p53. Phylogenetic analysis can be as simple as a multiple sequence alignment (MSA) where you assemble p53 protein sequences that you obtain from NCBI and perform an alignment using either NCBI tools or proprietary software. The BLAST feature of NCBI has a quick MSA feature. It can be a bit cumbersome, but it is a good place to start. Human p53 has a unique accession number (AAC12971.1) which you can use to do a protein BLAST search. At the top of the results page will appear an option for you to perform a multiple alignment. RNA DETECTION Question: How much p53 mRNA is expressed in prostate cancer cells and where is it localized during development? You know well that p53 has a tumor suppressor function and that its expression is eliminated or dramatically reduced in many cancers, so you want to determine the levels of p53 mRNA in a normal versus transformed human cell type. You extract RNA from prostate cancer cells (PC-3) and normal prostate cells (DU-145) and perform one of three types of analysis: Northern Blot. The tried and true method for RNA detection. Total RNA from cells is separated by size on an agarose gel, transferred to a membrane and that membrane is probed for the gene of interest using a radiolabeled piece of DNA or RNA that is complementary to your target. Labeled nucleic acids used in this manner are universally referred to as “probes.” Advantages: Quantitative, allows detection of multiple species using a single probe Disadvantages: Not as sensitive as RT-PCR, usually takes three days Reverse Transcription PCR (RT-PCR). For quick and dirty analysis of RNA expression. cDNA is synthesized from your purified RNA using oligo d(T) and reverse transcriptase. Primers corresponding to your gene of interest are used to amplify from the cDNA. If your p53 mRNA was abundant, you will get a strong band corresponding to your amplicon size. Advantages: Very quick - results same day Disadvantages: Not quantitative, prone to nonspecific amplification products that can be misleading. Quantitative (or real time) RT-PCR (qRT-PCR). What has become the gold standard for mRNA quantitation. In this case your cDNA is amplified in an instrument that detects fluorescence and you use a fluorescent “probe” that is an oligonucleotide that is complimentary to your target sequence. This probe only fluoresces when it is degraded during amplificaion, so tou then perform standard PCR using primers that flank the probe sequence and the more production of the amplicon, the higher the fluorescence. Advantages: Quantitative, Very quick - results same day Disadvantages: also prone to nonspecific amplification products that can be misleading - reproducibility must be carefully monitored In Situ Hybridization. If you become interested in the expression of p53 during development, you may want to find out which tissues contain mature mRNA. Many mRNAs are present but not translated during development, so in this instance it may be of interest to find out where p53 mRNA is made. If you were working in the Drosophila model system, you would take fresh embryos, fix and permeabilize them and then soak them in a buffer containing a fluorescent (or otherwise labeled) fragment of nucleic acid that is complementary to your target. This probe would then bind to your target mRNA and you would visualize using fluorescence microscopy. PROTEIN DETECTION Question: How much p53 protein is produced and is its production regulated? '''OK, so you now know that p53 mRNA is expressed in DU-145 but not PC-3 cells. You are not satisfied, however, because you know that it is p53 protein that is the important functional player, so you need to find out whether p53 protein is being made in DU-145. In this case, the PC-3 cells will serve as a negative control because you know they don’t make p53 mRNA so they can’t make p53 protein. '''Find an Antibody The only way to specifically detect the presence of an endogenous protein is by using an antibody. This is good and bad. Good because antibodies can be extremely sensitive and specific. Bad because antibodies can be extremely non-specific and not so sensitive. Your job is to find an extremely well validated antibody for your protein of interest. In the case of p53, your in luck because for a protein as well-studied as this, you can easily find publications that have done careful validations of commercial antibodies and you can just buy a good one. For the 20,000 or so proteins that haven’t been thoroughly studied this is a different matter. Be warned that there are a great many truly bad antibodies on the market. Of course you can always make your own (or have one made). It is useful to have recombinant p53 protein on hand to serve as a positive control for all of these assays. Western Blot. This is the current standard for protein detection. You make protein extracts from your PC-3 and DU-145 and resolve the proteins by size using SDS-PAGE. As with a Northern Blot, you then transfer your proteins to a membrane (usually nitrocellulose). You then incubate your membrane with your antibody that recognizes p53 protein. Then you incubate the membrane with an antibody that recognizes the antibody that recognizes p53 (this is called a secondary antibody). This secondary antibody is modified to possess an enzyme activity, usually horseradish peroxidase (HRP). You then incubate your membrane with chemicals that produce light in the presence of HRP, and you detect this light using x-ray film or a sensitive camera. The result, if you are lucky, is a band that migrates at the correct molecular mass and in your case is present in DU-145 but not PC-3 cells. Advantages: Can be semi-quantitative, sensitive Disadvantages: Extremely variable results depending on antibody quality and a myriad other factors. Reproducibility is often low, so the method is semi-quantitative at best. Immunoprecipitation. If the concentration of your protein is very low, you may need to purify it from a large quantity of cell extract. The easiest way to specifically purify p53 from cell extract is by immunoprecipitation where you physically attach your antibody to beads and incubate these antibody beads with your cell extract. All of the p53 in the cell extract will bind to the antibody, which since it is attached to beads will be easy to recover by centrifugation. You can then elute all of the proteins bound to the antibody with denaturing buffer and determine whether p53 is present by Western Blot. A typical negative control here would be the use of an antibody that doesn’t recognize p53. Capillary Electrophoresis. This method is being developed by a company called Protein Simple. It presents an interesting alternative to traditional Western Blots, but it requires the purchase of a pricey machine. PROTEIN LOCALIZATION Question: Where is p53 protein localized? '''So your western blot has verified that p53 protein is made in DU-145 cells, but you want to make sure that it can function as a transcription factor in these cells. One way to support this idea is to make sure that at least some of the protein is found in the nucleus. You need to perform sub-cellular localization. There are really only two methods with which to determine this: Immunohistochemistry. This method involves growing your DU-145 and PC-3 cells directly on microscope slides. When you have a sufficient number of cells grown on the slide, you can fix and permeablize the cells in order to keep everything stable and poke holes in the membranes in order to allow the antibody you are going to use to access all cellular compartments. In this case, the antibody you use will be fused to a fluorescent protein so that any interaction with p53 will be detectable by fluorescence microscopy. There are other ways to detect the position of the antibody, even including chemiluminescence, but fluorescence is arguably the easiest. In this case antibody validation is also extremely important. Nonspecific interactions should be ruled out by performing a control experiment in which the p53 antigen (usually a peptide) is included that will specifically soak up the antibody of choice. Under these conditions you’ll know that any fluorescent signal detected is NOT due to interaction with p53 because you’ve prevented that by having excess antigen present. In your case a control cell type can also be used since you know that PC-3 cells should have no signal. Differential Centrifugation. This is kind of an old fashioned term for a method that physically separates cells into their constituent organelles. If you want to know how much of p53 is cytoplasmic versus nuclear, you can physically separate nuceli from other cytoplasmic constituents and perform a Western blot on nuclear versus cytoplasmic extracts. This method can be extended to study other organelles, but the separation of other organelles is much trickier and requires finesse. TRANSCRIPTION AND CHROMATIN '''Question: Is p53 transcription regulated? Nuclear Run-On. All of the RNA detection assays described above simply measure the steady state levels of mRNA in a cell, which is the product of both transcription AND mRNA turnover. If you want to be sure that your gene is being actively transcribed (or not), you must use this method. Here you make nuclear extracts from your cells of interest and allow transcription to occur in the presence of radioactive nucleotides. This radioactive mixture of RNA is then used as a probe on a blot that contains your purified p53 DNA. It is almost like a reverse Northern. In the case of a Northern you are using a specific probe against a pool of heterogeneous mRNAs whereas here you are using heterogeneous RNA as a probe for a specific sequence. Note that you have to use DNA as the species on the blot because you need the presence of the complementary strand to bind to the RNA probe. Typically this would be a PCR product that you know corresponds to p53 sequence. Chromatin Immunoprecipitation (ChIP). ' '''Question: Is p53 transcription regulated by chromatin remodeling? '''Chromatin is generally considered to be a barrier to active transcription, thus it is often modified or “opened” in response to transcriptional activation. p53 expression is activated in response to DNA damage at the transcriptional level in part by histone remodeling. There are several methods used to analyze chromatin remodeling: 'DNAse I Hypersensitivity. Traditionally this would involve isolation of nuclei followed by limited digestion by DNAse I. DNA is then isolated and digested by restriction digestion as for a genomic Southern. Using your p53 promoter sequence as a probe you can compare DNAse I digested versus undigested DNA to see if any restriction fragments are cleaved under conditions where p53 activation would occur (e.g. DNA damage). Modern techniques expand on this idea by using [http://en.wikipedia.org/wiki/Next-generation_sequencing#Next-generation_methods Next''' Generation Sequencing.] In this case, the DNAse I products that are short (200-400 nt) are purified and identified by sequencing. The presence or absense of your gene(s) of interest is easily queried in the database of cleavage site that are obtained. This method is called DNAse-seq. '''ChIP-Seq. If you’d like to determine whether the p53 promoter is affected by histone modification, you can use an antibody that recognizes very specific histone modifications (e.g. Histone3 acetylation at Lys 56), then you can perform an immunoprecipitation on purified chromatin that has been crosslinked with formaldehyde and fragmented. The DNA that co-immunoprecipitates with the modified histone is identified by Next Gen Sequencing. Bisulfite Sequencing. One of the common epigenetic modifications is the methylation of DNA. In general, methylated DNA is associated with areas of higher transcription because it has the effect of “loosening” the chromatin. One can detect sites of DNA methylation by treating DNA with bisulfite. Treatment of DNA with bisulphite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. The DNA region of interest (i.e. the p53 promoter) is then sequenced to determine the extent to which C residues have been converted to U. mRNA TRANSLATION AND TURNOVER Question: Is the translation of p53 mRNA regulated? You (and many others) have become interested in how the growth factor known as Transforming Growth Factor β (TGFβ) affects p53 expression. When you applied TGFβ to your cells, you found that p53 mRNA levels increased but the protein level did not change. This is a classic initial indication that your gene of interested may be under translational control - i.e. the translation of the mRNA that is present in the cytosol is either specifically repressed or activated. To examine whether the translation of p53 mRNA is affected by TGFβ treatment you perform the following: Metabolic labeling. In this method you would simply introduce a radioactive amino acid (usually 35S methionine into your cell culture after TGFβ treatment. At several time points you “quench” the reaction with cold methionine and make total cell extract from which you would immunoprecipitate the p53 protein. Instead of detecting p53 by Western Blot, you expose your dried SDS-PAGE gel to x-ray film or a PhosphorImager screen in order to detect the amount of radiolabeled p53. If TGFβ is reducing p53 translation, then the amount of labeled p53 protein will go down after treatment. If you also detect total p53 by Western blot, you likely won’t see any difference in protein amounts since by metabolic labeling you are just looking at newly synthesized protein. Polysome analysis. mRNAs that are actively being translated are completely covered in ribosomes (ribosomes have a ~35 nt “footprint,” so a 3.5 kb mRNA will have 350 ribosomes on it). This mRNA/ribosome complex is called a polysome, and it just so happens to be one of the most dense things in the cell. Thus, one can separate actively translating mRNAs from those not being translated by centrifugation through a dense medium like sucrose in a method very similar to that described for differential centrifugation. If you find (by Northern Blot) that p53 mRNA is found among the less dense fractions of a sucrose sedimentation experiment when TGFβ is present, then you have good evidence that its translation is being reduced. Typical controls include the addition of known translation inhibitors (e.g. antibiotics) and the analysis of constitutively translated proteins like actin (although one must exercise care when choosing controls to study in cases where major signaling pathways are altered, as is the case for TGFβ, because even the controls can be regulated). Question: Is part of the upregulation of p53 mRNA in response to DNA damage due to decreased mRNA turnover (degradation)? ''' Since you’ve found the p53 mRNA levels actually increase upon TGFβ treatment, you assume that its rate of transcription has increased. However, you remember that the concentration of mRNA in a cell is actually the product of the transcription rate and the degradation or turnover rate. As such, you set out to use two methods to determine if the rate of p53 mRNA turnover decreases upon TGFβ treatment: '''Global Transcription Inhibition. You can use a drug such as α-amanatin, which is a potent inhibitor of Pol II transcription and then analyze p53 mRNA concentrations, by Northern Blot or qRT-PCR, over time. Since you’ve inhibited new synthesis, you will be measuring the rate at which p53 mRNA is turned over. This is usually done over a 12-24 hour time course. Your controls would be mRNAs that are known to have short and long half-lives. The obvious disadvantage to this is that you’ve severely altered cell function by shutting down transcription - the idea is that your decay time course is short enough to allow you to ignore these effects. An alternative method that gets around this problem is the use of the following: Repressible promoters. If you are worried about the side-effects of using a general transcription inhibitor, you can express p53 on a plasmid that carriers a repressible promoter, such as the Tet-off system which shuts down transcription from the tetracycline. The disadvantage here is that you are not analyzing endogenous p53, and your construct may lack important features that are required for regulation (e.g. the 5’ and 3’ UTRs). PROTEIN TURNOVER Question: Does the half-life of p53 protein change in response to DNA damage? Ubiquitination assay. Steady state levels of p53 are kept low in part by continuous proteasomal degradation. Upon activation (e.g. in response to DNA damage), p53 protein levels increase dramatically by inhibition of proteolysis. The degradation rate of p53 is governed by a balance of ubiquitination and de-ubiquitination, thus the amount of ubiquitin conjugated p53 is directly proportional to its degradation rate. In order to determine the amount of ubiquitination, one performs Western Blot analysis on cell lysates derived from cells either subjected to DNA damage or not (e.g. UV light). The blot is probed with anti-p53 antibody to determine the level of modification. p53 that is ubiquitinated will migrate more slowly. Importantly, proteasome activity must be inhibited in this experiment because ubiquitinated protein is rapidly degraded and thus won’t be detected in the absence of an inhibitor. Therefore, one should observe significantly more slowly migrating p53 (poly-ubiquitination usually results in a smear of high molecular weight bands) in cells that were not treated with UV compare to those that were. Protein Half Life Determination. Similar to an mRNA half-life determination, one can block new protein synthesis in cells with a translation inhibitor, typically cycloheximide. Cell lysates taken at time points after inhibition can be analyzed by Western Blot for p53 levels, which can be quantitated and plotted versus time to derive the time it takes to degrade half of the protein present at the initial time point. Inhibitor analysis. The identification of proteolytic pathways that may be involved in p53 degradation could be achieved by analyzing the sensitivity to inhibitors. Knowing that p53 is rapidly turned over during steady state conditions, one would simply screen a multitude of protease inhibitors in order, screening for those that increased p53 levels. In many cases, more than one candidate will be identified due to low inhibitor specificity or the involvement of more than one pathway. MACROMOLECULAR INTERACTIONS Question: Does p53 physically interact with other proteins? Co-immunoprecipitation. Considering that p53 responds to DNA damage, you predict that it might physically interact with a DNA damage sensing protein like CHEK1. To test this you can use a p53 antibody to immunoprecipitate, being careful to maintain conditions that preserve protein-protein interactions (e.g. lower salt, mild detergent). When you perform a Western Blot analysis of the stuff that immunoprecipitated you can probe the blot with an anti-CHEK1 antibody to see if it is there. This assay is prone to high background, so don’t forget your controls! Another important consideration is stoichiometry...it is important to keep track of how much p53 is brought down relative to the amount of its binding partner. If the stoichiometric ratio is low, then the interaction is more likely to be nonspecific. Question: What DNA sequences does p53 bind? ChIP-Seq. Here you would perform exactly the same type of ChIP-Seq experiment described above, but this time you would use an anti-p53 antibody to pull down protein/DNA complexes. You identify the sequences bound by Next Generation Sequencing. Electrophoretic Mobility Shift Assay (EMSA). This assay is useful mainly for the study of known DNA protein interactions. In the case of P53, if you want to determine the minimal piece of DNA that is required for binding, you could synthesize fragments of DNA, radioactively label them and incubated them with recombinant p53 protein. In order to analyze complexes, you separate bound from unbound DNA on a non-denaturing polyacrylamide gel. This type of electrophoresis allows you to maintain complexes. You should see a significant lowering of the migration rate for DNA that is bound by p53. The best control here is a mutant version of p53 that you know lacks DNA binding activity. Question: Are there proteins that bind to the p53 3’ UTR that might regulate translation or turnover? RNA affinity chromatography. If you were interested in determining the mechanism by which p53 translation is regulated, you might start by analyzing the function of the 5’ and 3’ UTRs. In many cases regulatory proteins bind to these regions of the mRNA and thereby control either turnover, translation or both. PROTEIN PURIFICATION Question: Does recombinant p53 have the same DNA binding properties as endogenous protein? Expression Systems. You are ready to do some serious biochemical analysis of p53 function in vitro so you’ll need lots of protein...and it needs to retain all of its functions. You will therefore screen a variety of expression systems to find out which gives the best yield of active protein. The most common expression systems are bacterial (E.coli), insect (Sf-9), or mammalian (HeLa). There are many factors that go into choosing the best expression system. E coli is by far the most common because it is the least expensive. If you need your protein to be modified, however, then you must use a eukaryotic system. Also, in many cases, proteins are not soluble in bacteria, so optimized conditions or alternative systems must be employed. More recently, in vitro protein production systems allow the synthesis of large quantities of protein in cell lysates, usually E coli, but they are very expensive. Protein Tags. In order to purify your recombinant p53 protein, you will need to add a sequence tag that you will use to detect and purify your protein. By far the most common tag is a series of N or C-terminally placed His residues (His-tag), but other tags include small peptides (FLAG or Myc) or larger proteins (e.g. GST). The choice of tag, and position of tag, must be determined empirically. Affinity Purification. Once you’ve generated a strain of E coli that expresses His-tagged p53, you must then purifiy the protein away from the multitude of bacterial proteins that are also present. For a His-tag, one takes advantage of the fact that His residues in tandem bind with high affinity to immobilized nickel. So in this case you would pass your E coli lysate over a nickel column, wash it extensively then elute your protein with the Histidine analogue, imidazole. Generally this yields fairly pure protein, but often other types of protein chromatography are required if highly pure protein is needed. Latest activity Photos and videos are a great way to add visuals to your wiki. Find videos about your topic by exploring Wikia's Video Library. Category:Browse Category:Education